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Resistance to insulin and insulin-like growth factor 1 (IGF1) in pancreatic β-cells causes overt diabetes in mice; thus, therapies that sensitize β-cells to insulin may protect patients with diabetes against β-cell failure1–3. Here we identify an inhibitor of insulin receptor (INSR) and IGF1 receptor (IGF1R) signalling in mouse β-cells, which we name the insulin inhibitory receptor (inceptor; encoded by the gene Iir). Inceptor contains an extracellular cysteine-rich domain with similarities to INSR and IGF1R4, and a mannose 6-phosphate receptor domain that is also found in the IGF2 receptor (IGF2R)5. Knockout mice that lack inceptor (Iir−/−) exhibit signs of hyperinsulinaemia and hypoglycaemia, and die within a few hours of birth. Molecular and cellular analyses of embryonic and postnatal pancreases from Iir−/− mice showed an increase in the activation of INSR–IGF1R in Iir−/− pancreatic tissue, resulting in an increase in the proliferation and mass of β-cells. Similarly, inducible β-cell-specific Iir−/− knockout in adult mice and in ex vivo islets led to an increase in the activation of INSR–IGF1R and increased proliferation of β-cells, resulting in improved glucose tolerance in vivo. Mechanistically, inceptor interacts with INSR–IGF1R to facilitate clathrin-mediated endocytosis for receptor desensitization. Blocking this physical interaction using monoclonal antibodies against the extracellular domain of inceptor resulted in the retention of inceptor and INSR at the plasma membrane to sustain the activation of INSR–IGF1R in β-cells. Together, our findings show that inceptor shields insulin-producing β-cells from constitutive pathway activation, and identify inceptor as a potential molecular target for INSR–IGF1R sensitization and diabetes therapy. The insulin inhibitory receptor (inceptor) is identified as a negative regulator of insulin and IGF1 signalling that could be targeted for β-cell regeneration in treatments for diabetes.

BACKGROUND AND AIMS: NAFLD is initiated by steatosis and can progress through fibrosis and cirrhosis to HCC. The RNA binding protein human antigen R (HuR) controls RNAs at the posttranscriptional level; hepatocyte HuR has been implicated in the regulation of diet-induced hepatic steatosis. The present study aimed to understand the role of hepatocyte HuR in NAFLD development and progression to fibrosis and HCC. APPROACH AND RESULTS: Hepatocyte-specific, HuR-deficient mice and control HuR-sufficient mice were fed either a normal diet or an NAFLD-inducing diet. Hepatic lipid accumulation, inflammation, fibrosis, and HCC development were studied by histology, flow cytometry, quantitative PCR, and RNA sequencing. The liver lipidome was characterized by lipidomics analysis, and the HuR-RNA interactions in the liver were mapped by RNA immunoprecipitation sequencing. Hepatocyte-specific, HuR-deficient mice displayed spontaneous hepatic steatosis and fibrosis predisposition compared to control HuR-sufficient mice. On an NAFLD-inducing diet, hepatocyte-specific HuR deficiency resulted in exacerbated inflammation, fibrosis, and HCC-like tumor development. A multi-omic approach, including lipidomics, transcriptomics, and RNA immunoprecipitation sequencing revealed that HuR orchestrates a protective network of hepatic-metabolic and lipid homeostasis-maintaining pathways. Consistently, HuR-deficient livers accumulated, already at steady state, a triglyceride signature resembling that of NAFLD livers. Moreover, up-regulation of secreted phosphoprotein 1 expression mediated, at least partially, fibrosis development in hepatocyte-specific HuR deficiency on an NAFLD-inducing diet, as shown by experiments using antibody blockade of osteopontin. CONCLUSIONS: HuR is a gatekeeper of liver homeostasis, preventing NAFLD-related fibrosis and HCC, suggesting that the HuR-dependent network could be exploited therapeutically., Hepatology, 75 (4), ISSN:0270-9139, ISSN:1527-3350

Objective The liver regulates the availability of insulin to other tissues and is the first line insulin response organ physiologically exposed to higher insulin concentrations than the periphery. Basal insulin during fasting inhibits hepatic gluconeogenesis and glycogenolysis, whereas postprandial insulin peaks stimulate glycogen synthesis. The molecular consequences of chronic insulin deficiency for the liver have not been studied systematically. Methods We analyzed liver samples of a genetically diabetic pig model (MIDY) and of wild-type (WT) littermate controls by RNA sequencing, proteomics, and targeted metabolomics/lipidomics. Results Cross-omics analyses revealed increased activities in amino acid metabolism, oxidation of fatty acids, ketogenesis, and gluconeogenesis in the MIDY samples. In particular, the concentrations of the ketogenic enzyme 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2) and of retinol dehydrogenase 16 (RDH16), which catalyzes the first step in retinoic acid biogenesis, were highly increased. Accordingly, elevated levels of retinoic acid, which stimulates the expression of the gluconeogenic enzyme phosphoenolpyruvate carboxykinase (PCK1), were measured in the MIDY samples. In contrast, pathways related to extracellular matrix and inflammation/pathogen defense response were less active than in the WT samples. Conclusions The first multi-omics study of a clinically relevant diabetic large animal model revealed molecular signatures and key drivers of functional alterations of the liver in insulin-deficient diabetes mellitus. The multi-omics data set provides a valuable resource for comparative analyses with other experimental or clinical data sets., Highlights • MIDY pigs were used to study consequences of insulin-deficient diabetes for the liver. • RDH16 and HMGCS2 were drivers of stimulated gluconeogenesis and ketogenesis in MIDY pigs. • Hepatic immune functions and extracellular matrix were reduced in MIDY pigs. • This multi-omics data resource is valuable for analyses of other liver omics data sets.

Inflammation and cellular stress mutually regulate each other and are hallmarks of various diseases. The mechanistic basis of this interaction remains incompletely understood. Here, we report that proteostatic stress triggers the transcription and intracellular accumulation of Dickkopf1 (DKK1) in a p38-dependent fashion. Intracellular DKK1 sustains the high inflammatory tone in inherently stressed cells and is required for mounting cytokine responses following ligation of toll-like and cytokine receptors. Leveraging unbiased proteomics and bioinformatic analysis, we identify a physical interaction between intracellular DKK1 and Copine1 (CPNE1), which promotes NFκB-dependent inflammation irrespective of upstream phosphorylation checkpoints and IκBα degradation. Genetic deletion of DKK1, but not pharmacological neutralization of secreted DKK1, ameliorates inflammation and organ damage in a mouse model of sepsis. Collectively, we identify DKK1 as a target of the cellular stress response and uncover a non-canonical, cell-intrinsic function of DKK1 in the control of NFκB-mediated inflammation, thus providing novel insights into the coupling of homeostatic perturbations and immunity.

ObjectiveAdrenocortical hormone levels increase in obesity, potentially contributing to development of obesity-associated pathologies. Here we explored whether lipidomic remodeling of the adrenal gland could mediate altered adrenocortical steroidogenesis during obesity.MethodsLipidomic analysis was performed in adrenal glands using shotgun mass spectrometry (MS), and steroid profiling of sera by liquid chromatography tandem mass spectrometry (LC-MS/MS) from lean and obese mice. Gene expression analysis was performed in adrenal glands and adrenocortical cell populations. The role of Fatty Acid Desaturase 2 (FADS2) and arachidonic acid on steroid hormone production was studied in primary adrenal gland cell cultures.ResultsAdrenal glands of obese mice displayed a distinct lipidomic profile, encompassing longer and more unsaturated storage lipids and phospholipids compared to adrenal glands of lean mice. Arachidonoyl acyl chains were abundant in the adrenal gland phospholipidome and increased upon obesity. This was accompanied by increasedFads2expression, the rate-limiting enzyme of arachidonic acid synthesis, and enhanced plasma adrenocortical hormone levels. Inhibition of FADS2 in primary adrenal gland cell cultures abolished steroidogenesis, which was restored by arachidonic acid supplementation.ConclusionsOur data suggest that the FADS2 – arachidonic acid axis regulates adrenocortical hormone synthesis, while alterations in the content of arachidonoyl chains in the adrenal gland phopsholipidome could account for disturbed adrenocortical hormone production.HighlightsThe adrenal gland lipidome is remodeled in obesity.Arachidonoyl groups are abundant in the adrenal gland phospholipidome and increase in obesity.FADS2 is highly expressed in the adrenal gland and its expression is further increased in obesity.FADS2 inhibition blunts adrenocortical steroidogenesis in primary adrenal gland cell cultures, while arachidonic acid supplementation restores it.

Pancreatic islet beta cells employ secretory granules for the storage and glucose-stimulated release of the hormone insulin. The competence of an insulin granule for exocytosis depends on spatial and temporal variables such as its proximity to the plasma membrane as well as its age, with newly-generated granules being preferentially released. The molecular underpinnings for the control of these variables remain largely unknown and their uncovering is of high relevance for the study of diabetes, which results from deficient insulin secretion. However, we still lack a comprehensive view about the molecular composition of the insulin granules and how this may change over their lifetime. Here we report a strategy for the background-free purification of insulin secretory granules of distinct age from insulinoma INS-1 cells. We show that utilization of an immuno-based affinity approach for pulse-chase labeled insulin secretory granules, produces a highly enriched granular fraction. Our approach precludes typical contaminants from the solid phase and may be designed to purify secretory granules of a distinct age.

A calorie-rich diet is one reason for the continuous spread of metabolic syndromes in western societies. Smart food design is one powerful tool to prevent metabolic stress, and the search for suitable bioactive additives is a continuous task. The nutrient-sensing insulin signaling pathway is an evolutionary conserved mechanism that plays an important role in metabolism, growth and development. Recently, lipid cues capable to stimulate insulin signaling were identified. However, the mechanistic base of their activity remains obscure to date. Here, we show that specific AKT/ Protein kinase B isoforms are responsive to dietary lipid extract compositions and potentiate cellular insulin signaling levels. Our data add a new dimension to existing models and position Drosophila as a powerful model to study the relation between dietary lipid cues and the insulin induced cellular signal cascade.

Objective Shotgun lipidomics enables an extensive analysis of lipids from tissues and fluids. Each specimen requires appropriate extraction and processing procedures to ensure good coverage and reproducible quantification of the lipidome. Adipose tissue (AT) has become a research focus with regard to its involvement in obesity-related pathologies. However, the quantification of the AT lipidome is particularly challenging due to the predominance of triacylglycerides, which elicit high ion suppression of the remaining lipid classes. Methods We present a new and validated method for shotgun lipidomics of AT, which tailors the lipid extraction procedure to the target specimen and features high reproducibility with a linear dynamic range of at least 4 orders of magnitude for all lipid classes. Results Utilizing this method, we observed tissue-specific and diet-related differences in three AT types (brown, gonadal, inguinal subcutaneous) from lean and obese mice. Brown AT exhibited a distinct lipidomic profile with the greatest lipid class diversity and responded to high-fat diet by altering its lipid composition, which shifted towards that of white AT. Moreover, diet-induced obesity promoted an overall remodeling of the lipidome, where all three AT types featured a significant increase in longer and more unsaturated triacylglyceride and phospholipid species. Conclusions The here presented method facilitates reproducible systematic lipidomic profiling of AT and could be integrated with further –omics approaches used in (pre-) clinical research, in order to advance the understanding of the molecular metabolic dynamics involved in the pathogenesis of obesity-associated disorders., Graphical abstract Image 1, Highlights • Validated shotgun lipidomics method of AT covering 300 lipids of 20 classes and linear dynamic range of 4 orders of magnitude. • Increase of longer and more unsaturated triacylglycerides and phospholipids in brown and white AT under high-fat diet. • Differences in the lipidomes of gonadal, subcutaneous and brown AT.

Resolution of inflammation is essential for tissue homeostasis and a promising approach to inflammatory disorders. Here we found that DEL-1, a secreted protein inhibiting leukocyte-endothelial adhesion and inflammation initiation, also functions as a non-redundant downstream effector in inflammation clearance. In human and murine periodontitis, waning of inflammation correlated with DEL-1 upregulation, whereas resolution of experimental periodontitis failed in DEL-1 deficiency. This concept was mechanistically substantiated in acute monosodium urate crystal-induced inflammation, where the pro-resolution function of DEL-1 was attributed to effective apoptotic neutrophil clearance (efferocytosis). DEL-1-mediated efferocytosis induced liver-X-receptor-dependent macrophage reprogramming to pro-resolving phenotype and was required for optimal production of at least certain specific pro-resolving mediators. Experiments in transgenic mice with cell-specific overexpression of DEL-1 linked its anti-leukocyte recruitment action to endothelial-derived DEL-1 and its efferocytic/pro-resolving action to macrophage-derived DEL-1. Thus, the compartmentalized expression of DEL-1 facilitates distinct homeostatic functions in an appropriate context that can be harnessed therapeutically., This work was supported by grants from the European Research Council (DEMETINL to T.C.), the Deutsche Forschungsgemeinschaft (Transregio-SFB 83 to Ü.C. as well as Transregio-SFB 127 and Transregio-SFB 205 to T.C.) and the NIH (AI068730, DE024153, DE024716, DE026152 and DE015254 to G.H.). I.K. was supported by the MeDDriveGrant (60.400) from the Technische Universität (TU) Dresden Medical Faculty. A.Ca was supported by Spanish MINECO SAF2014–56819R and SAF2015–71878-REDT. This work was supported in part by intramural NIDCR, NIH funding.

Summary Trained innate immunity fosters a sustained favorable response of myeloid cells to a secondary challenge, despite their short lifespan in circulation. We thus hypothesized that trained immunity acts via modulation of hematopoietic stem and progenitor cells (HSPCs). Administration of β-glucan (prototypical trained-immunity-inducing agonist) to mice induced expansion of progenitors of the myeloid lineage, which was associated with elevated signaling by innate immune mediators, such as IL-1β and granulocyte-macrophage colony-stimulating factor (GM-CSF), and with adaptations in glucose metabolism and cholesterol biosynthesis. The trained-immunity-related increase in myelopoiesis resulted in a beneficial response to secondary LPS challenge and protection from chemotherapy-induced myelosuppression in mice. Therefore, modulation of myeloid progenitors in the bone marrow is an integral component of trained immunity, which to date, was considered to involve functional changes of mature myeloid cells in the periphery., Graphical Abstract, Highlights • Trained immunity (TI) modulates hematopoietic progenitors in bone marrow • TI is associated with adaptations in cell metabolism in progenitors • TI increases expansion of hematopoietic progenitors and myelopoiesis • TI promotes beneficial responses to systemic inflammation and chemotherapy, Modulation of hematopoietic stem and progenitor cells during trained immunity allows a sustained response of myeloid cells to a secondary challenge despite their short lifespan in circulation.

The liver regulates the availability of insulin to other tissues and is the first line insulin response organ being physiologically exposed to higher insulin concentrations than the periphery. Basal insulin during fasting inhibits hepatic gluconeogenesis and glycogenolysis, whereas postprandial insulin peaks stimulate glycogen synthesis. The molecular consequences of chronic insulin deficiency for the liver have not been studied systematically. We analyzed liver samples of a genetically diabetic pig model (MIDY) and of wild-type (WT) littermate controls by RNA sequencing, proteomics and targeted metabolomics/lipidomics. Cross-omics analyses revealed increased activity in gluconeogenesis, ketogenesis, amino acid metabolism and oxidation of fatty acids in the MIDY samples, whereas pathways related to extracellular matrix and inflammation/pathogen defense response were less active than in the WT samples. Our study identified key drivers of these alterations, such as RDH16 and HMGCS2 for stimulated gluconeogenesis and ketogenesis, in a clinically relevant large animal model for insulin-deficient diabetes mellitus.

Blood is arguably the most important bodily fluid and its analysis provides crucial health status information. A first routine measure to narrow down diagnosis in clinical practice is the differential blood count, determining the frequency of all major blood cells. What is lacking to advance initial blood diagnostics is an unbiased and quick functional assessment of blood that can narrow down the diagnosis and generate specific hypotheses. To address this need, we introduce the continuous, cell-by-cell morpho-rheological (MORE) analysis of diluted whole blood, without labeling, enrichment or separation, at rates of 1000 cells/sec. In a drop of blood we can identify all major blood cells and characterize their pathological changes in several disease conditions in vitro and in patient samples. This approach takes previous results of mechanical studies on specifically isolated blood cells to the level of application directly in blood and adds a functional dimension to conventional blood analysis., eLife digest When you are sick and go to the doctor, it is often not obvious what exactly is wrong — what is causing fever, nausea, shortness of breath or other symptoms. It is important to find this out quickly so that the right action can be taken. One of the first steps is to obtain a blood sample and to count how many of the different blood cells are present in it. This is called a complete blood count, and the information it provides has turned out to be surprisingly useful. A large number of certain white blood cells, for example, can show that the body is fighting an infection. But there might be several reasons why the number of white blood cells has increased, so this information alone is often not enough for a specific diagnosis. There are many hundreds of possible tests that can supplement the results of a complete blood count. These might identify bacteria or measure the concentrations of certain molecules in the blood, for example. But which test will give the important clue that reveals the source of the illness? This can be difficult to predict. Although each test helps to narrow down the final diagnosis they become increasingly expensive and time-consuming to perform, and rapid action is often important when treating a disease. Can we get more decisive information from the initial blood test by measuring other properties of the blood cells? Toepfner et al. now show that this is possible using a technique called real-time deformability cytometry. This method forces the blood cells in a small drop of blood to flow extremely rapidly through a narrow microfluidic channel while they are imaged by a fast camera. A computer algorithm can then analyze the size and stiffness of the blood cells in real-time. Toepfner et al. show that this approach can detect characteristic changes that affect blood cells as a result of malaria, spherocytosis, bacterial and viral infections, and leukemia. Furthermore, many thousands of blood cells can be measured in a few minutes — fast enough to be suitable as a diagnostic test. These proof-of-concept findings can now be used to develop actual diagnostic tests for a wide range of blood-related diseases. The approach could also be used to test which of several drugs is working to treat a certain medical condition, and to monitor whether the treatment is progressing as planned.

Using glycosylated full-length human insulin receptor reconstituted into lipid nanodiscs, we show that insulin binding to the dimeric receptor converts its ectodomains from an inverted U-shaped to a T-shaped conformation. This unprecedented structural rearrangement of the ectodomains propagates to the transmembrane domains, which are well separated in the inactive conformation, but come together upon insulin binding, allowing autophosphorylation of the cytoplasmic kinase domains.

Blood is arguably the most important bodily fluid and its analysis provides crucial health status information. A first routine measure to narrow down diagnosis in clinical practice is the differential blood count, determining the frequency of all major blood cells. What is lacking to advance initial blood diagnostics is an unbiased and quick functional assessment of blood that can narrow down the diagnosis and generate specific hypotheses. To address this need, we introduce the continuous, cell-by-cell morpho-rheological (MORE) analysis of whole blood, without labeling, enrichment or separation, at rates of 1,000 cells/sec. In a drop of blood we can identify all major blood cells and characterize their pathological changes in several disease conditions in vitro and in patient samples. This approach takes previous results of mechanical studies on specifically isolated blood cells to the level of application directly in whole blood and adds a functional dimension to conventional blood analysis.

Driven by interactions between lipids and proteins, biological membranes display lateral heterogeneity that manifests itself in a mosaic of liquid-ordered (Lo) or raft, and liquid-disordered (Ld) or non-raft domains with a wide range of different properties and compositions. In giant plasma membrane vesicles and giant unilamellar vesicles, specific binding of Cholera Toxin (CTxB) to GM1 glycolipids is a commonly used strategy to label raft domains or Lo membrane environments. However, these studies often use acyl-chain labeled bodipy-GM1 (bdGM1), whose headgroup accessibility and membrane order or phase partitioning may differ from those of GM1, rendering the interpretation of CTxB binding data quite problematic. To unravel the molecular basis of CTxB binding to GM1 and bdGM1, we explored the partitioning and the headgroup presentation of these gangliosides in the Lo and Ld phases using atomistic molecular dynamics simulations complemented by CTxB binding experiments. The conformation of both GM1 and bdGM1 was shown to be largely similar in the Lo and Ld phases. However, bdGM1 showed reduction in receptor availability when reconstituted into synthetic bilayer mixtures, highlighting that membrane phase partitioning of the gangliosides plays a considerable role in CTxB binding. Our results suggest that the CTxB binding is predominately modulated by the partitioning of the receptor to an appropriate membrane phase. Further, given that the Lo and Ld partitioning of bdGM1 differs from those of GM1, usage of bdGM1 for studying GM1 behavior in cells can lead to invalid interpretation of experimental data. publishedVersion

Objective The prevalence of diabetes mellitus and associated complications is steadily increasing. As a resource for studying systemic consequences of chronic insulin insufficiency and hyperglycemia, we established a comprehensive biobank of long-term diabetic INSC94Y transgenic pigs, a model of mutant INS gene-induced diabetes of youth (MIDY), and of wild-type (WT) littermates. Methods Female MIDY pigs (n = 4) were maintained with suboptimal insulin treatment for 2 years, together with female WT littermates (n = 5). Plasma insulin, C-peptide and glucagon levels were regularly determined using specific immunoassays. In addition, clinical chemical, targeted metabolomics, and lipidomics analyses were performed. At age 2 years, all pigs were euthanized, necropsied, and a broad spectrum of tissues was taken by systematic uniform random sampling procedures. Total beta cell volume was determined by stereological methods. A pilot proteome analysis of pancreas, liver, and kidney cortex was performed by label free proteomics. Results MIDY pigs had elevated fasting plasma glucose and fructosamine concentrations, C-peptide levels that decreased with age and were undetectable at 2 years, and an 82% reduced total beta cell volume compared to WT. Plasma glucagon and beta hydroxybutyrate levels of MIDY pigs were chronically elevated, reflecting hallmarks of poorly controlled diabetes in humans. In total, ∼1900 samples of different body fluids (blood, serum, plasma, urine, cerebrospinal fluid, and synovial fluid) as well as ∼17,000 samples from ∼50 different tissues and organs were preserved to facilitate a plethora of morphological and molecular analyses. Principal component analyses of plasma targeted metabolomics and lipidomics data and of proteome profiles from pancreas, liver, and kidney cortex clearly separated MIDY and WT samples. Conclusions The broad spectrum of well-defined biosamples in the Munich MIDY Pig Biobank that will be available to the scientific community provides a unique resource for systematic studies of organ crosstalk in diabetes in a multi-organ, multi-omics dimension., Molecular Metabolism, 6 (8)

Lateral compositional and physicochemical heterogeneity is a ubiquitous feature of cellular membranes on various length scales, from molecular assemblies to micrometric domains. Segregated lipid domains of increased local order, referred to as rafts, are believed to be prominent features in eukaryotic plasma membranes; however, their exact nature (i.e. size, lifetime, composition, homogeneity) in live cells remains difficult to define. Here we present evidence that both synthetic and natural plasma membranes assume a wide range of lipid packing states with varying levels of molecular order. These states may be adapted and specifically tuned by cells during active cellular processes, as we show for stimulated insulin secretion. Most importantly, these states regulate both the partitioning of molecules between coexisting domains and the bioactivity of their constituent molecules, which we demonstrate for the ligand binding activity of the glycosphingolipid receptor GM1. These results confirm the complexity and flexibility of lipid-mediated membrane organization and reveal mechanisms by which this flexibility could be functionalized by cells.

The epidermal growth factor receptor (EGFR) regulates several critical cellular processes and is an important target for cancer therapy. In lieu of a crystallographic structure of the complete receptor, atomistic molecular dynamics (MD) simulations have recently shown that they can excel in studies of the full-length receptor. Here we present atomistic MD simulations of the monomeric N-glycosylated human EGFR in biomimetic lipid bilayers that are, in parallel, also used for the reconstitution of full-length receptors. This combination enabled us to experimentally validate our simulations, using ligand binding assays and antibodies to monitor the conformational properties of the receptor reconstituted into membranes. We find that N-glycosylation is a critical determinant of EGFR conformation, and specifically the orientation of the EGFR ectodomain relative to the membrane. In the absence of a structure for full-length, posttranslationally modified membrane receptors, our approach offers new means to structurally define and experimentally validate functional properties of cell surface receptors in biomimetic membrane environments.

Laurdan Identifies Different Lipid Membranes in Eukaryotic Cells Enrico Gratton and Michelle A. Digman CONTENTS 13.1 Introduction 283 13.1.1 Spectroscopic Properties of Laurdan 283 13.2 The Phasor Approach to Spectral and Lifetime Analysis 287 13.3 The Lifetime Phasor Transformation and Its Interpretation 289 13.4 Results of the Analysis of the Emission of Laurdan Using Spectral and Lifetime Phasors in GUVs Model Systems 291 13.5 The Lifetime Phasor for Laurdan in GUVs 292 13.6 Live Cell Membrane Fluidity 295 13.6.1 Spectral Phasors 295 13.6.2 Lifetime Phasors in Live 3T3 Cells 297 13.7 Conclusions and Further Considerations 299 13.8 Methods 300 13.8.1 Preparation of the GUVs 300 13.8.2 NIH3T3 Cell Cultures 301 13.8.3 FLIM Analysis 301 13.8.4 Spectral Analysis 301 Acknowledgment 301 References 302 13.1 INTRODUCTION 13.1.1 S pectroscopic P roperties of L aurdan There are several commonly used approaches for the study of membrane properties of live cells based on fluorescence probes. In one approach, lipids with specific fluores- cent markers are incorporated in the cell membranes. The advantage of this approach is that it is possible to study the membrane distribution of specific lipids. However, when the aim of the study is to detect membrane microdomains independently of their